Construction method and detection method of cigar leaf fingerprint and application thereof

Abstract

The application discloses a method for constructing a fingerprint of cigar tobacco leaves, a method for detecting the fingerprint and application of the fingerprint. The method for constructing the fingerprint of the cigar tobacco leaves comprises the following steps: mixing a cigar tobacco leaf powder with a solvent, performing ultrasonic extraction, performing solid-liquid separation, collecting a liquid, and preparing a sample solution; performing ultra-high performance liquid chromatography tandem mass spectrometry on the sample solution to construct the fingerprint of the cigar tobacco leaves; wherein the chromatographic conditions of the ultra-high performance liquid chromatography tandem mass spectrometry comprise that a mobile phase comprises mobile phase A and mobile phase B, the mobile phase A is a 0.1% formic acid aqueous solution, the mobile phase B is a 0.1% formic acid acetonitrile solution, and an elution mode is gradient elution. The fingerprint of the cigar tobacco leaves is constructed by using the ultra-high performance liquid chromatography tandem mass spectrometry, and the overall chemical composition of the cigar tobacco leaves can be comprehensively, objectively and stably characterized, thereby providing a scientific basis for research on comprehensive evaluation of the origin, variety and quality of the cigar tobacco leaves.

Description

Technical Field

[0001] This application belongs to the field of detection and analysis technology, and relates to the method for constructing and detecting fingerprint spectra of cigar tobacco leaves and their applications. Background Technology

[0002] Cigar tobacco leaves (Nicotiana tabacum L.) are tobacco leaves from a dicotyledonous plant in the Solanaceae family, specifically used for making cigars. They are usually from specific varieties of tobacco and undergo unique processes such as drying, fermentation, and aging to develop the unique flavor, aroma, and burning characteristics of cigars.

[0003] The quality of cigar tobacco leaves is closely related to their origin ecology and varietal genetic characteristics. Subtle but crucial differences in chemical composition exist between tobacco leaves from different sources, directly determining the final aroma, taste, and burning quality of the cigar. Traditionally, the identification and quality evaluation of cigar tobacco leaves have relied primarily on sensory evaluation and the testing of a few routine chemical indicators. However, sensory evaluation is easily influenced by the subjective experience, physiological state, and environmental factors of the evaluators, resulting in poor repeatability and a lack of quantitative standards. Routine chemical testing can only obtain the content of single components such as total sugar, total nitrogen, and nicotine, failing to comprehensively reflect the overall characteristics of the complex chemical composition of the tobacco leaves. With the increasing demand in the global cigar market for authenticity, quality stability, and traceability of high-end products, traditional methods of identifying cigar tobacco leaves are insufficient to effectively address issues such as adulteration, varietal confusion, and quality grading. Therefore, it is necessary to establish a method that can comprehensively, objectively, and stably characterize the overall chemical composition of cigar tobacco leaves, enabling accurate identification of tobacco leaf origin, variety, and quality grade. Summary of the Invention

[0004] Based on this, some embodiments provide methods for constructing and detecting fingerprint spectra of cigar tobacco leaves and their applications.

[0005] In some embodiments, a method for constructing a fingerprint spectrum of cigar tobacco leaves is provided, comprising the following steps:

[0006] Cigar tobacco leaves were mixed with a solvent, subjected to ultrasonic extraction, solid-liquid separation, and the liquid was collected to prepare a test solution.

[0007] The test solution was subjected to ultra-high performance liquid chromatography-tandem mass spectrometry to construct the fingerprint spectrum of the cigar tobacco leaves;

[0008] The chromatographic conditions for the ultra-high performance liquid chromatography-tandem mass spectrometry include: the mobile phase comprises mobile phase A and mobile phase B.

[0009] The mobile phase A is a 0.1% formic acid aqueous solution, and the mobile phase B is a 0.1% formic acid acetonitrile solution. The elution method is gradient elution.

[0010] The elution gradient of the mobile phase is as follows: -3 min to 0 min, column equilibration, mobile phase B volume percentage 0%, mobile phase A volume percentage 100%; 0 min to 4 min, mobile phase B volume percentage 0%, mobile phase A volume percentage 100%; 4 min to 8 min, mobile phase B volume percentage changes from 0% to 25%, mobile phase A volume percentage changes from 100% to 75%; 8 min to 14 min, mobile phase B volume percentage changes from 25% to 65%, mobile phase A volume percentage changes from 75% to 35%; 14 min to 28 min, mobile phase B volume percentage changes from 65% to 100%, mobile phase A volume percentage changes from 35% to 0%; 28 min to 32 min, mobile phase B volume percentage 100%, mobile phase A volume percentage 0%.

[0011] In some embodiments, the provided method for constructing fingerprint profiles of cigar tobacco leaves includes ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) chromatographic conditions comprising one or more of the following:

[0012] (1) The column temperature is 38℃~42℃;

[0013] (2) The flow rate is 0.2 mL / min to 0.25 mL / min;

[0014] (3) The injection volume is 1 μL to 1.5 μL;

[0015] (4) The chromatographic column was Waters ACQUITY UPLC HSS T3.

[0016] In some embodiments, the provided method for constructing fingerprint profiles of cigar tobacco leaves includes ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) conditions comprising one or more of the following:

[0017] (1) The ion source is an H-ESI source;

[0018] (2) Positive and negative ion detection modes are adopted. The spray voltage for positive ionization mode is 3000V~4000V, and the spray voltage for negative ionization mode is 2000V~3000V.

[0019] (3) The collision gas is nitrogen;

[0020] (4) The sheath gas is nitrogen, the sheath gas flow rate is 40 Arb~50 Arb, and the sheath gas temperature is 300℃~350℃;

[0021] (5) The auxiliary gas is nitrogen, and the flow rate of the auxiliary gas is 10 Arb to 15 Arb;

[0022] (6) The sweeping gas velocity is 0Arb;

[0023] (7) The temperature of the ion transport tube is 300℃~350℃;

[0024] (8) The temperature of the vaporization chamber is 250℃~300℃;

[0025] (9) Data collection time is 30-35 minutes;

[0026] (10) The scanning range of the first-order mass number is 100~1200 m / z;

[0027] (11) The resolution of the first-level full scan is 60,000;

[0028] (12) The secondary resolution is 60000;

[0029] (13) The radio frequency voltage of the S lens is set to 50%~55%;

[0030] (14) The expected LC peak width is 6S~7S;

[0031] (15) Acquisition speed: 15 scans / s ~ 18 scans / s;

[0032] (16) In MS / MS mode, the collision energy is set to 20eV, 40eV and 80eV respectively.

[0033] In some implementations, the provided method for constructing fingerprint profiles of cigar tobacco leaves satisfies one or more of the following conditions:

[0034] (1) The mass-to-volume ratio of the cigar tobacco leaves to the solvent is 0.5 g: (10~15) mL;

[0035] (2) The solvent includes a methanol aqueous solution with a volume fraction of 70% to 80%;

[0036] (3) The temperature for ultrasonic extraction is 25℃~35℃, and the extraction time is 30min~35min;

[0037] (4) Solid-liquid separation is carried out by centrifugation, wherein the centrifugation is performed at 8000r / min~12000r / min for 5min~10min.

[0038] In some embodiments, the provided method for constructing a fingerprint spectrum of cigar tobacco leaves includes a fingerprint spectrum with 74 characteristic peaks, the substances corresponding to the characteristic peaks comprising:

[0039] L-Threonic acid, allantoin, D-(-)-quinic acid, methylsuccinic acid, D-(+)-malic acid, isocitrate, succinic acid, pantothenic acid, acetylpropionic acid, pentenoic acid, caffeic acid, 3-methylsalicylic acid, azelaic acid, (15Z)-9,12,13-trihydroxy-15-octadecadienoic acid, 12-oxooctadecadienoic acid, arachidic acid, (±)-13-hydroxy-9,11-octadecadienoic acid, ethyl linolenic acid, 13(S)-hydroxy Octadectotrienoic acid, 16-hydroxyhexadecanoic acid, L-aspartic acid, L-valine, proline, N-acetyl-DL-glutamic acid, 4-oxoproline, L-aspartic acid benzyl ester, N-phenylacetyl-L-aspartic acid, N-acetyl-L-phenylalanine, indole-3-lactic acid, indole-3-acetyl-L-aspartic acid, N-acetyl-DL-tryptophan, tryptophan, DL-stachyine, trigonelline, (S)-nicotine Nicotine-N-oxide, N-methylnicotinamide, trans-3-indoleacrylic acid, cotinine, 5-methoxy-N,N-diisopropyltryptamine, lorinolide, asiatic acid, isostevimol, ligustrol A, perilla lactone, betulin, oleanolic acid, catechol, protocatechuic acid, aesculin, rutin, hyperoside, quercetin, kaempferol, choline, mannitol, 2-amino-1,3,4-octadecanetriol, β-carotene, L-glucose L-lactone, L-ribose-1,4-lactone, (3R)-4,4-dimethyl-2-oxotetrahydrofuran-3-ylβ-D-glucopyranoside, limonamide, aurantamide, docosahexaenoic acid ethanolamide, hexadecylamide, 1,7-bis(4-hydroxyphenyl)-5-methoxyheptane-3-one, phenylheptanone, hypoxanthine, xanthine nucleoside, p-coumaraldehyde, coenzyme Q2, porphyrin, 3-aminoquinoline, 8-hydroxyquinoline.

[0040] In some embodiments, a method for detecting cigar tobacco leaves is provided, comprising the following steps:

[0041] The cigar tobacco leaves to be tested were mixed with a solvent and subjected to ultrasonic extraction to prepare the sample solution to be tested.

[0042] The sample solution to be tested was detected by ultra-high performance liquid chromatography-tandem mass spectrometry to obtain a spectrum;

[0043] The spectrum was compared and analyzed with the fingerprint spectrum of cigar tobacco constructed by the method described above;

[0044] The chromatographic conditions for the ultra-high performance liquid chromatography-tandem mass spectrometry include: the mobile phase comprises mobile phase A and mobile phase B.

[0045] The mobile phase A is a 0.1% formic acid aqueous solution, and the mobile phase B is a 0.1% formic acid acetonitrile solution. The elution method is gradient elution.

[0046] The elution gradient of mobile phase B is as follows: -3 min to 0 min, column equilibration, mobile phase B volume percentage is 0%; 0 min to 4 min, mobile phase B volume percentage is 0%; 4 min to 8 min, mobile phase B volume percentage changes from 0% to 25%; 8 min to 14 min, mobile phase B volume percentage changes from 25% to 65%; 14 min to 28 min, mobile phase B volume percentage changes from 65% to 100%; 28 min to 32 min, mobile phase B volume percentage is 100%.

[0047] In some embodiments, the provided method for detecting cigar tobacco leaves includes ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) chromatographic conditions comprising one or more of the following:

[0048] (1) The column temperature is 38℃~42℃;

[0049] (2) The flow rate is 0.2 mL / min to 0.25 mL / min;

[0050] (3) The injection volume is 1 μL to 1.5 μL;

[0051] (4) The chromatographic column was Waters ACQUITY UPLC HSS T3.

[0052] In some embodiments, the provided method for detecting cigar tobacco leaves includes ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) conditions comprising one or more of the following:

[0053] (1) The ion source is an H-ESI source;

[0054] (2) Positive and negative ion detection modes are adopted. The spray voltage for positive ionization mode is 3000V~4000V, and the spray voltage for negative ionization mode is 2000V~3000V.

[0055] (3) The collision gas is nitrogen;

[0056] (4) The sheath gas is nitrogen, the sheath gas flow rate is 40 Arb~50 Arb, and the sheath gas temperature is 300℃~350℃;

[0057] (5) The auxiliary gas is nitrogen, and the flow rate of the auxiliary gas is 10 Arb to 15 Arb;

[0058] (6) The sweeping gas velocity is 0Arb;

[0059] (7) The temperature of the ion transport tube is 300℃~350℃;

[0060] (8) The temperature of the vaporization chamber is 250℃~300℃;

[0061] (9) Data collection time is 30-35 minutes;

[0062] (10) The scanning range of the first-order mass number is 100~1200 m / z;

[0063] (11) The resolution of the first-level full scan is 60,000;

[0064] (12) The secondary resolution is 60000;

[0065] (13) The radio frequency voltage of the S lens is set to 50%~55%;

[0066] (14) The expected LC peak width is 6S~7S;

[0067] (15) Acquisition speed: 15 scans / s ~ 18 scans / s;

[0068] (16) In MS / MS mode, the collision energy is set to 20eV, 40eV and 80eV respectively.

[0069] In some embodiments, the provided method for detecting cigar tobacco leaves satisfies one or more of the following conditions:

[0070] (1) The mass-to-volume ratio of the cigar tobacco leaves to the solvent is 0.5 g: (10~15) mL;

[0071] (2) The solvent includes a methanol aqueous solution with a volume fraction of 70% to 80%;

[0072] (3) The temperature for ultrasonic extraction is 25℃~35℃, and the extraction time is 30min~35min;

[0073] (4) Solid-liquid separation is carried out by centrifugation, wherein the centrifugation is performed at 8000r / min~12000r / min for 5min~10min.

[0074] In some embodiments, the application of the fingerprint pattern construction method for cigar tobacco leaves or the detection method for cigar tobacco leaves includes at least one of the following applications:

[0075] (1) Application in tracing the origin of cigar tobacco leaves;

[0076] (2) Application in the quality control of cigar tobacco leaves;

[0077] (3) Application in the identification of cigar tobacco varieties.

[0078] A fingerprint spectrum of cigar tobacco leaves was constructed using ultra-high performance liquid chromatography-tandem mass spectrometry, which can comprehensively, objectively, and stably characterize the overall chemical composition of cigar tobacco leaves, providing a scientific basis for the study of the origin, variety, and comprehensive quality evaluation of cigar tobacco leaves. Attached Figure Description

[0079] To more clearly illustrate the technical solutions in the embodiments and examples of this application, and to more completely understand this application and its beneficial effects, the accompanying drawings used in the description of the embodiments or examples will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of this application. Those skilled in the art can obtain other drawings based on these drawings without any creative effort.

[0080] Figure 1 These are ion chromatograms for different extraction methods, where A is the positive ion mode and B is the negative ion mode.

[0081] Figure 2 The images show ion chromatograms for different extraction solvents, where A is the positive ion mode and B is the negative ion mode.

[0082] Figure 3 The images show ion chromatograms at different extraction temperatures, where A is the positive ion pattern and B is the negative ion pattern.

[0083] Figure 4 The images show ion chromatograms at different extraction times, where A is the positive ion pattern and B is the negative ion pattern.

[0084] Figure 5 The images show ion chromatograms for different feed-to-liquid ratios, where A is the positive ion mode and B is the negative ion mode.

[0085] Figure 6 These are ion chromatograms under different purification conditions, where A is the positive ion pattern and B is the negative ion pattern.

[0086] Figure 7 The images show ion chromatograms for different mobile phase systems, where A is the positive ion mode and B is the negative ion mode.

[0087] Figure 8 The images show ion chromatograms for different columns. In the images, A is the positive ion mode chromatogram, B is the negative ion mode chromatogram, peak 1 is (R)-nicotine, peak 2 is (S)-nicotine, peak 3 is L-malic acid, and peak 4 is 4-oxoproline.

[0088] Figure 9 These are ion chromatograms at different column temperatures, where A is the positive ion mode and B is the negative ion mode.

[0089] Figure 10 The images show ion chromatograms for different injection volumes, where A is the positive ion mode and B is the negative ion mode.

[0090] Figure 11 These are ion chromatograms at different flow rates, where A is the positive ion pattern and B is the negative ion pattern.

[0091] Figure 12 The images show ion chromatograms for different elution gradients, where A is the positive ion pattern and B is the negative ion pattern.

[0092] Figure 13 The images show mass spectra at different collision energies, where A is the mass spectrum at collision energy E1, B is the mass spectrum at collision energy E2, and C is the mass spectrum at collision energy E3.

[0093] Figure 14 Bar charts showing data scans at different acquisition speeds are provided. A is a bar chart showing data scans at an acquisition speed of 10 scans / s, and B is a bar chart showing data scans at an acquisition speed of 15 scans / s.

[0094] Figure 15 A pie chart showing metabolite categories;

[0095] Figure 16 The image shows the fingerprint spectrum of cigar tobacco leaves, where A is the positive ion pattern and B is the negative ion pattern.

[0096] Figure 17 PCA score chart for cigar tobacco leaves;

[0097] Figure 18 LDA score chart for cigar tobacco leaves;

[0098] Figure 19 Score plot for PLS-DA model of cigar tobacco leaves;

[0099] Figure 20 This is a PLS-DA permutation test plot, where "permutations" represents "random permutations" and "components" represents "components".

[0100] Figures 1-20 The “Time” mentioned here refers to “time”, “Relative Abundance” refers to “relative abundance”, and “Colored according to classes in M1” means “different colors in the figure represent different groups in model M1”. Detailed Implementation

[0101] To facilitate understanding of this application, a more complete description will be provided below with reference to the accompanying drawings. Preferred embodiments of this application are shown in the drawings. However, this application can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of this application.

[0102] It should be noted that when a component is said to be "fixed to" another component, it can be directly attached to the other component or there may be an intervening component. When a component is said to be "connected to" another component, it can be directly connected to the other component or there may be an intervening component.

[0103] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0104] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0105] The terms "and / or," "or / and," and "and / or" as used in this application encompass any one of two or more related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected using at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that in this application, the technical solution undoubtedly includes solutions connected by "logical AND," and also undoubtedly includes solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and "a combination of A and B."

[0106] In this application, the terms "multiple", "various", "multiple times", "multi-dimensional", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more than or equal to two.

[0107] The terms “combinations thereof,” “any combination thereof,” and “any combination thereof” as used in this application include all suitable combinations of any two or more of the listed items.

[0108] In this application, the term "suitable" as used in "suitable combination", "suitable method", "any suitable method", etc., refers to the ability to implement the technical solution of this application, solve the technical problem of this application, and achieve the expected technical effect of this application.

[0109] In this application, terms such as "preferred," "better," "more suitable," and "ideal" are merely used to describe implementation methods or embodiments that achieve better results, and should be understood not to limit the scope of protection of this application.

[0110] In this application, terms such as "further," "even further," and "particularly" are used to describe purposes and indicate differences in content, but should not be construed as limiting the scope of protection of this application.

[0111] In this application, "optionally," "optionally," and "optional" mean that something is optional, that is, it means that it is selected from either "with" or "without." If there are multiple "optional" entries in a technical solution, unless otherwise specified, and there are no contradictions or mutual constraints, each "optional" entry shall be independent.

[0112] In this application, the technical features described in an open-ended manner include both closed technical solutions consisting of the listed features and open technical solutions that include the listed features.

[0113] In this application, numerical intervals (i.e., numerical ranges) are involved. Unless otherwise specified, the selected numerical distributions within the aforementioned numerical intervals are considered continuous and include the two endpoints (i.e., the minimum and maximum values) of the numerical range, as well as every value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints. In this document, this is equivalent to directly listing every integer. For example, if t is an integer selected from 1 to 10, it means that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Furthermore, when multiple ranges are provided to describe features or characteristics, these ranges can be merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges to which they are included.

[0114] Unless otherwise specified, the temperature parameters in this application are permitted to be either constant-temperature treatment or variations within a certain temperature range. It should be understood that the constant-temperature treatment allows temperature fluctuations within the precision range of the instrument control, such as ±5℃, ±4℃, ±3℃, ±2℃, or ±1℃.

[0115] In this application, % (w / w) and wt% both represent weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass-volume percentage.

[0116] In this application, "room temperature" generally refers to 5℃~30℃, and more preferably 25±5℃.

[0117] In some embodiments, a method for constructing a fingerprint spectrum of cigar tobacco leaves is provided, comprising the following steps:

[0118] Cigar tobacco leaves were mixed with a solvent, subjected to ultrasonic extraction, solid-liquid separation, and the liquid was collected to prepare a test solution.

[0119] The test solution was subjected to ultra-high performance liquid chromatography-tandem mass spectrometry to construct the fingerprint spectrum of the cigar tobacco leaves;

[0120] The chromatographic conditions for the ultra-high performance liquid chromatography-tandem mass spectrometry include: the mobile phase comprises mobile phase A and mobile phase B.

[0121] The mobile phase A is a 0.1% formic acid aqueous solution, and the mobile phase B is a 0.1% formic acid acetonitrile solution. The elution method is gradient elution.

[0122] The elution gradient of the mobile phase is as follows: -3 min to 0 min, column equilibration, mobile phase B volume percentage 0%, mobile phase A volume percentage 100%; 0 min to 4 min, mobile phase B volume percentage 0%, mobile phase A volume percentage 100%; 4 min to 8 min, mobile phase B volume percentage changes from 0% to 25%, mobile phase A volume percentage changes from 100% to 75%; 8 min to 14 min, mobile phase B volume percentage changes from 25% to 65%, mobile phase A volume percentage changes from 75% to 35%; 14 min to 28 min, mobile phase B volume percentage changes from 65% to 100%, mobile phase A volume percentage changes from 35% to 0%; 28 min to 32 min, mobile phase B volume percentage 100%, mobile phase A volume percentage 0%.

[0123] In some embodiments, the provided method for constructing fingerprint profiles of cigar tobacco leaves includes ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) chromatographic conditions such as a column temperature of 38°C to 42°C, for example, 38°C, 39°C, 40°C, 41°C, 42°C, etc., or any range of two of the aforementioned values.

[0124] In some embodiments, the provided method for constructing fingerprint profiles of cigar tobacco leaves includes ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) with chromatographic conditions including a flow rate of 0.2 mL / min to 0.25 mL / min.

[0125] In some embodiments, the provided method for constructing fingerprint profiles of cigar tobacco leaves includes ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) with chromatographic conditions including an injection volume of 1 μL to 1.5 μL.

[0126] In some embodiments, the provided method for constructing fingerprint profiles of cigar tobacco leaves includes ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) with the following chromatographic conditions: a Waters ACQUITY UPLC HSS T3 column.

[0127] In some embodiments, the provided method for constructing fingerprint profiles of cigar tobacco leaves includes ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) conditions including an H-ESI ion source.

[0128] In some embodiments, the provided method for constructing fingerprint profiles of cigar tobacco leaves includes the following mass spectrometry conditions for ultra-high performance liquid chromatography-tandem mass spectrometry: positive and negative ion detection modes, positive ionization mode spray voltage of 3000V~4000V, and negative ionization mode spray voltage of 2000V~3000V.

[0129] In some embodiments, the provided method for constructing fingerprint profiles of cigar tobacco leaves includes ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) with the following mass spectrometry conditions: the collision gas is nitrogen.

[0130] In some embodiments, the provided method for constructing fingerprint profiles of cigar tobacco leaves includes ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) with the following mass spectrometry conditions: the sheath gas is nitrogen, the sheath gas flow rate is 40 Arb to 50 Arb, and the sheath gas temperature is 300°C to 350°C.

[0131] In some embodiments, the provided method for constructing fingerprint profiles of cigar tobacco leaves includes ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) with the following mass spectrometry conditions: nitrogen as the auxiliary gas and an auxiliary gas flow rate of 10 Arb to 15 Arb.

[0132] In some embodiments, the provided method for constructing fingerprint profiles of cigar tobacco leaves includes ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) with the following mass spectrometry conditions: a sweep gas flow rate of 0 Arb.

[0133] In some embodiments, the provided method for constructing fingerprint profiles of cigar tobacco leaves includes ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) with the following mass spectrometry conditions: ion transfer tube temperature of 300°C to 350°C.

[0134] In some embodiments, the provided method for constructing fingerprint profiles of cigar tobacco leaves includes ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) with the following mass spectrometry conditions: vaporization chamber temperature 250°C to 300°C.

[0135] In some embodiments, the provided method for constructing fingerprint profiles of cigar tobacco leaves includes ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) with a data acquisition time of 30-35 minutes.

[0136] In some embodiments, the provided method for constructing fingerprint profiles of cigar tobacco leaves includes ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) with mass spectrometry conditions including a first-order mass number scan range of 100-1200 m / z.

[0137] In some embodiments, the provided method for constructing fingerprint profiles of cigar tobacco leaves includes ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) with the following mass spectrometry conditions: a first-stage full scan resolution of 60,000.

[0138] In some embodiments, the provided method for constructing fingerprint profiles of cigar tobacco leaves includes ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) with the following mass spectrometry conditions: a secondary resolution of 60,000.

[0139] In some embodiments, the provided method for constructing fingerprint profiles of cigar tobacco leaves includes ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) with the following mass spectrometry conditions: the radio frequency voltage of the S-lens is set to 50%~55%.

[0140] In some embodiments, the provided method for constructing fingerprint profiles of cigar tobacco leaves includes ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) conditions including: an expected LC peak width of 6S to 7S.

[0141] In some embodiments, the provided method for constructing fingerprint profiles of cigar tobacco leaves includes ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) with mass spectrometry conditions including an acquisition rate of 15 scans / s to 18 scans / s.

[0142] In some embodiments, the provided method for constructing fingerprint profiles of cigar tobacco leaves includes the following mass spectrometry conditions for ultra-high performance liquid chromatography-tandem mass spectrometry: in MS / MS mode, the collision energy is set to 20 eV, 40 eV, and 80 eV, respectively.

[0143] In some embodiments, the provided method for constructing fingerprint profiles of cigar tobacco leaves uses a mass-to-volume ratio of cigar tobacco leaves to solvent of 0.5 g: (10~15) mL, for example, 0.5 g: 10 mL, 0.5 g: 11 mL, 0.5 g: 12 mL, 0.5 g: 13 mL, 0.5 g: 14 mL, 0.5 g: 15 mL, etc., or any range of the aforementioned two ratios.

[0144] In some embodiments, the provided method for constructing fingerprint profiles of cigar tobacco leaves includes a solvent comprising a methanol aqueous solution with a volume fraction of 70% to 80%. For example, the volume fraction of the methanol aqueous solution can be 70%, 75%, 80%, or any range of the aforementioned two values.

[0145] In some embodiments, the provided method for constructing fingerprint profiles of cigar tobacco leaves involves ultrasonic extraction at a temperature of 25°C to 35°C and an extraction time of 30 to 35 minutes. For example, the ultrasonic extraction temperature can be 25°C, 30°C, 35°C, or any range of two of the aforementioned values; the ultrasonic extraction time can be 30 minutes, 31 minutes, 32 minutes, 33 minutes, 34 minutes, 35 minutes, or any range of two of the aforementioned values.

[0146] In some embodiments, the provided method for constructing fingerprint profiles of cigar tobacco leaves employs centrifugation for solid-liquid separation, wherein the centrifugation is performed at 8000 r / min to 12000 r / min for 5 min to 10 min.

[0147] In some embodiments, the provided method for constructing a fingerprint spectrum of cigar tobacco leaves includes a fingerprint spectrum with 74 characteristic peaks, the substances corresponding to the characteristic peaks comprising:

[0148] L-Threonic acid, allantoin, D-(-)-quinic acid, methylsuccinic acid, D-(+)-malic acid, isocitrate, succinic acid, pantothenic acid, acetylpropionic acid, pentenoic acid, caffeic acid, 3-methylsalicylic acid, azelaic acid, (15Z)-9,12,13-trihydroxy-15-octadecadienoic acid, 12-oxooctadecadienoic acid, arachidic acid, (±)-13-hydroxy-9,11-octadecadienoic acid, ethyl linolenic acid, 13(S)-hydroxy Octadectotrienoic acid, 16-hydroxyhexadecanoic acid, L-aspartic acid, L-valine, proline, N-acetyl-DL-glutamic acid, 4-oxoproline, L-aspartic acid benzyl ester, N-phenylacetyl-L-aspartic acid, N-acetyl-L-phenylalanine, indole-3-lactic acid, indole-3-acetyl-L-aspartic acid, N-acetyl-DL-tryptophan, tryptophan, DL-stachyine, trigonelline, (S)-nicotine Nicotine-N-oxide, N-methylnicotinamide, trans-3-indoleacrylic acid, cotinine, 5-methoxy-N,N-diisopropyltryptamine, lorinolide, asiatic acid, isostevimol, ligustrol A, perilla lactone, betulin, oleanolic acid, catechol, protocatechuic acid, aesculin, rutin, hyperoside, quercetin, kaempferol, choline, mannitol, 2-amino-1,3,4-octadecanetriol, β-carotene, L-glucose L-lactone, L-ribose-1,4-lactone, (3R)-4,4-dimethyl-2-oxotetrahydrofuran-3-ylβ-D-glucopyranoside, limonamide, aurantamide, docosahexaenoic acid ethanolamide, hexadecylamide, 1,7-bis(4-hydroxyphenyl)-5-methoxyheptane-3-one, phenylheptanone, hypoxanthine, xanthine nucleoside, p-coumaraldehyde, coenzyme Q2, porphyrin, 3-aminoquinoline, 8-hydroxyquinoline.

[0149] In some embodiments, a method for detecting cigar tobacco leaves is provided, comprising the following steps:

[0150] The cigar tobacco leaves to be tested were mixed with a solvent and subjected to ultrasonic extraction to prepare the sample solution to be tested.

[0151] The sample solution to be tested was detected by ultra-high performance liquid chromatography-tandem mass spectrometry to obtain a spectrum;

[0152] The spectrum was compared and analyzed with the fingerprint spectrum of cigar tobacco constructed by the method described above;

[0153] The chromatographic conditions for the ultra-high performance liquid chromatography-tandem mass spectrometry include: the mobile phase comprises mobile phase A and mobile phase B.

[0154] The mobile phase A is a 0.1% formic acid aqueous solution, and the mobile phase B is a 0.1% formic acid acetonitrile solution. The elution method is gradient elution.

[0155] The elution gradient of mobile phase B is as follows: -3 min to 0 min, column equilibration, mobile phase B volume percentage is 0%; 0 min to 4 min, mobile phase B volume percentage is 0%; 4 min to 8 min, mobile phase B volume percentage changes from 0% to 25%; 8 min to 14 min, mobile phase B volume percentage changes from 25% to 65%; 14 min to 28 min, mobile phase B volume percentage changes from 65% to 100%; 28 min to 32 min, mobile phase B volume percentage is 100%.

[0156] In some embodiments, the chromatographic conditions of the ultra-high performance liquid chromatography-tandem mass spectrometry method for detecting cigar tobacco leaves include: a column temperature of 38°C to 42°C, for example, 38°C, 39°C, 40°C, 41°C, 42°C, etc., or any range of two of the aforementioned values.

[0157] In some embodiments, the provided method for detecting cigar tobacco leaves includes ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) with chromatographic conditions of a flow rate of 0.2 mL / min to 0.25 mL / min.

[0158] In some embodiments, the provided method for detecting cigar tobacco leaves includes ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) with chromatographic conditions including an injection volume of 1 μL to 1.5 μL.

[0159] In some embodiments, the provided method for detecting cigar tobacco leaves includes chromatographic conditions for ultra-high performance liquid chromatography-tandem mass spectrometry, such that the chromatographic column is a Waters ACQUITY UPLC HSS T3.

[0160] In some embodiments, the provided method for detecting cigar tobacco leaves includes ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) with the following mass spectrometry conditions: the ion source is an H-ESI source.

[0161] In some embodiments, the provided method for detecting cigar tobacco leaves includes the following mass spectrometry conditions for ultra-high performance liquid chromatography-tandem mass spectrometry: positive and negative ion detection modes, positive ionization mode spray voltage of 3000V~4000V, and negative ionization mode spray voltage of 2000V~3000V.

[0162] In some embodiments, the provided method for detecting cigar tobacco leaves includes ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) with the following mass spectrometry conditions: the collision gas is nitrogen.

[0163] In some embodiments, the provided method for detecting cigar tobacco leaves includes the following mass spectrometry conditions for ultra-high performance liquid chromatography-tandem mass spectrometry: the sheath gas is nitrogen, the sheath gas flow rate is 40 Arb to 50 Arb, and the sheath gas temperature is 300°C to 350°C.

[0164] In some embodiments, the provided method for detecting cigar tobacco leaves includes the following mass spectrometry conditions for ultra-high performance liquid chromatography-tandem mass spectrometry: the auxiliary gas is nitrogen, and the auxiliary gas flow rate is 10 Arb to 15 Arb.

[0165] In some embodiments, the provided method for detecting cigar tobacco leaves includes ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) with the following mass spectrometry conditions: a sweep gas flow rate of 0 Arb.

[0166] In some embodiments, the provided method for detecting cigar tobacco leaves includes ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) with the following mass spectrometry conditions: ion transfer tube temperature of 300°C to 350°C.

[0167] In some embodiments, the provided method for detecting cigar tobacco leaves includes ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) with the following mass spectrometry conditions: vaporization chamber temperature 250°C to 300°C.

[0168] In some embodiments, the provided method for detecting cigar tobacco leaves includes ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) with the following mass spectrometry conditions: data acquisition time of 30-35 minutes.

[0169] In some embodiments, the provided method for detecting cigar tobacco leaves includes ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) with mass spectrometry conditions including a first-order mass number scan range of 100-1200 m / z.

[0170] In some embodiments, the provided method for detecting cigar tobacco leaves includes ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) with the following mass spectrometry conditions: a first-stage full scan resolution of 60,000.

[0171] In some embodiments, the provided method for detecting cigar tobacco leaves includes ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) with the following mass spectrometry conditions: a secondary resolution of 60,000.

[0172] In some embodiments, the provided method for detecting cigar tobacco leaves includes ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) with the following mass spectrometry conditions: the radio frequency voltage of the S-lens is set to 50%~55%.

[0173] In some embodiments, the provided method for detecting cigar tobacco leaves includes ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) with the following mass spectrometry conditions: an expected LC peak width of 6S to 7S.

[0174] In some embodiments, the provided method for detecting cigar tobacco leaves includes ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) with mass spectrometry conditions including a sampling rate of 15 scans / s to 18 scans / s.

[0175] In some embodiments, the provided method for detecting cigar tobacco leaves includes the following mass spectrometry conditions for ultra-high performance liquid chromatography-tandem mass spectrometry: in MS / MS mode, the collision energy is set to 20 eV, 40 eV, and 80 eV, respectively.

[0176] In some embodiments, the provided method for detecting cigar tobacco leaves satisfies one or more of the following conditions:

[0177] In some embodiments, the provided method for detecting cigar tobacco leaves has a mass-to-volume ratio of 0.5 g to (10-15) mL of cigar tobacco leaves to solvent.

[0178] In some embodiments, the provided method for detecting cigar tobacco leaves includes a solvent comprising an aqueous methanol solution with a volume fraction of 70% to 80%.

[0179] In some embodiments, the provided method for detecting cigar tobacco leaves involves ultrasonic extraction at a temperature of 25°C to 35°C for a time of 30 to 35 minutes.

[0180] In some embodiments, the provided method for detecting cigar tobacco leaves employs centrifugation for solid-liquid separation, wherein centrifugation is performed at 8000 r / min to 12000 r / min for 5 min to 10 min.

[0181] In some embodiments, the fingerprint pattern construction method of the cigar tobacco leaves or the detection method of the cigar tobacco leaves are provided for application in the traceability of the origin of cigar tobacco leaves.

[0182] In some embodiments, the fingerprint pattern construction method or the detection method of the cigar tobacco leaves is provided for application in the quality control of cigar tobacco leaves.

[0183] In some embodiments, the fingerprint pattern construction method of the cigar tobacco leaves or the detection method of the cigar tobacco leaves are provided for application in the identification of cigar tobacco leaf varieties.

[0184] The following are specific embodiments. They are intended to provide a more detailed description of this application to help those skilled in the art and researchers better understand it. The technical conditions described do not constitute any limitation on this application. Any modifications made within the scope of the claims of this application are protected by the claims.

[0185] Unless otherwise stated, all raw materials and reagents used in the following examples are commercially available or can be prepared by known methods. Experimental methods not specifying particular conditions in the examples were performed under conventional conditions, such as those described in literature, books, or methods recommended by the manufacturer.

[0186] Example 1: Establishment of a metabolomics method for cigar tobacco leaves

[0187] 1.1 Experimental Materials and Equipment

[0188] Raw material: Cigar tobacco leaves, produced in Sichuan.

[0189] Reagents: Formic acid (chromatographic grade), Chengdu Kelong Chemical Co., Ltd.; Ethyl acetate, Chengdu Kelong Chemical Co., Ltd.; Chromatographic grade methanol, Thermo Fisher Scientific, USA; Chromatographic grade acetonitrile, Thermo Fisher Scientific, USA.

[0190] Instruments and Equipment: JL-4500B Multifunctional Grinding Machine, Yongkang Sufeng Industry and Trade Co., Ltd.; SHB-Ⅲ Circulating Water Multipurpose Vacuum Pump, Zhengzhou Great Wall Science and Industry Trade Co., Ltd.; SB25-12DTD Ultrasonic Cleaner, Ningbo Xinzhi Biotechnology Co., Ltd.; HD-3000 Vortex Strainer, Hangzhou Youning Instrument Co., Ltd.; Milli-Q DIRECT 8 Ultrapure Water System, Shanghai Yixing Electromechanical Equipment Co., Ltd.; H17.5R High-Speed ​​Refrigerated Centrifuge, Shanghai Luxiangyi Centrifuge Instrument Co., Ltd.; HZY-224 / 323 Electronic Balance, Huazhi (Fujian) Electronic Technology Co., Ltd.; Vanquish™ UHPLC Ultra-High Performance Liquid Chromatograph, Thermo Fisher Scientific, USA; Orbitrap Exploris 480 High Resolution Mass Spectrometer, Thermo Fisher Scientific, USA.

[0191] 1.2 Experimental Methods

[0192] 1.2.1 Optimization of Cigar Tobacco Leaf Pretreatment

[0193] 1.2.1.1 Optimization of Extraction Method

[0194] Ultrasonic extraction method: Accurately weigh 0.500g of cigar tobacco powder, place it in a 50mL centrifuge tube, add 10mL of pure methanol solution, and ultrasonically extract at 30℃ for 30min. After the extracted sample cools to room temperature, centrifuge at 10000r / min for 6min, take the supernatant, filter it through a 0.22μm microporous membrane, and set aside for later use.

[0195] Vortex extraction method: Accurately weigh 0.500g of cigar tobacco powder, place it in a 50mL centrifuge tube, add 10mL of pure methanol solution, vortex for 60s, centrifuge at 10000r / min for 6min, take the supernatant, filter it through a 0.22μm microporous membrane, and set aside for later use.

[0196] 1.2.1.2 Optimization of Extraction Solvent

[0197] The effects of four solvents—ethyl acetate, pure methanol, 75% methanol, and 50% methanol—on the extraction efficiency were investigated to determine the optimal extraction solvent.

[0198] 1.2.1.3 Optimization of Extraction Temperature

[0199] The effects of extraction temperatures of 20℃, 30℃, and 40℃ on the extraction effect were investigated to determine the optimal extraction temperature.

[0200] 1.2.1.4 Optimization of Extraction Time

[0201] The effects of ultrasonic extraction for 20 min, 30 min, 45 min, and 60 min on the extraction effect were investigated to determine the optimal extraction time.

[0202] 1.2.1.5 Optimization of the feed-liquid ratio

[0203] The effects of material-to-liquid ratios of 1:20 and 1:10 on the extraction effect were investigated to determine the optimal material-to-liquid ratio.

[0204] 1.2.1.6 Optimization of Concentration Conditions

[0205] Centrifugation: After the extracted sample is cooled to room temperature, it is centrifuged at 10000 r / min for 6 min and then filtered through a 0.22 μm microporous membrane.

[0206] Rotary concentration: Take 6 mL of the supernatant of the extract into the rotating flask of the rotary evaporator, evaporate to dryness at 40°C, add 3 mL of pure methanol, sonicate at 20°C for 5 min, remove the liquid and filter it through a 0.22 μm microporous membrane.

[0207] 1.2.2 Optimization of chromatographic conditions

[0208] 1.2.2.1 Optimization of the mobile phase

[0209] Three mobile phase systems were investigated: methanol-0.1% formic acid, acetonitrile-0.1% formic acid, and 0.1% formic acid-acetonitrile-0.1% formic acid. The optimal mobile phase system was determined based on factors such as the number of peaks, peak intensity, separation degree, and baseline stability.

[0210] 1.2.2.2 Optimization of Chromatographic Columns

[0211] Among commonly used chromatographic columns, the ACQUITY UPLC BEH C18 (2.1 mm × 50 mm, 1.7 μm) and ACQUITY UPLC HSS T3 (2.1 mm × 100 mm, 1.8 μm) columns were selected for evaluation. The effectiveness of the BEH C18 and T3 columns in compound separation was investigated to determine the optimal column.

[0212] 1.2.2.3 Optimization of column temperature

[0213] The peak elution and separation performance at column temperatures of 25℃, 30℃, and 40℃ were investigated to determine the optimal column temperature.

[0214] 1.2.2.4 Optimization of Injection Volume

[0215] The injection volume directly affects the separation effect of compounds. Here, the peak elution was investigated when the injection volume was 0.5 μL, 1.0 μL, and 2.0 μL, respectively, to determine the optimal injection volume.

[0216] 1.2.2.5 Flow rate optimization

[0217] The flow rates of 0.2 mL / min, 0.3 mL / min, and 0.4 mL / min were investigated, and the optimal flow rate of the mobile phase was selected based on the number of chromatographic peaks and the separation results.

[0218] 1.2.2.6 Gradient Optimization

[0219] The following five elution gradients were examined, as shown in Tables 1 to 5. The optimal elution gradient was selected based on the separation effect of different elution gradients.

[0220] Table 1 Elution gradient 1

[0221]

[0222] Table 2 Elution gradient 2

[0223]

[0224] Table 3 Elution gradient 3

[0225]

[0226] Table 4 Elution gradient 4

[0227]

[0228] Table 5 Elution gradient 5

[0229]

[0230] 1.2.3 Optimization of mass spectrometry conditions

[0231] The Exploris 480 is a high-performance, highly stable, ultra-high-resolution mass spectrometer with fast scanning speeds and powerful quantification capabilities. It efficiently processes complex samples, performing in-depth qualitative and quantitative analysis, and is designed for demanding research fields such as proteomics, metabolomics, and lipidomics. Therefore, recommended parameters for each omics are available in mass spectrometry. Here, to further optimize the collision energy and acquisition speed to improve the quantity and accuracy of metabolite identification, we present our findings.

[0232] 1.2.3.1 Optimization of Collision Energy

[0233] Here, we investigate three collision energies: E1 (20, 40, 60), E2 (30, 50, 150), and E3 (20, 40, 80) to select the optimal collision energy for the degree of fragmentation of the same compound.

[0234] 1.2.3.2 Optimization of Acquisition Speed

[0235] Two acquisition rates, 10 scans / s and 15 scans / s, were examined. By comparing the number of compounds, the optimal scheme was finally selected.

[0236] 1.3 Experimental Results and Analysis

[0237] 1.3.1 Optimization of Cigar Tobacco Leaf Pretreatment

[0238] 1.3.1.1 Optimization of Extraction Method

[0239] By comparing the total ion chromatograms of samples obtained under different extraction methods, ultrasonic extraction was determined to be the optimal method. Figure 1 The number of chromatographic peaks and the response intensity obtained by ultrasonic extraction are higher than those obtained by vortex extraction. Therefore, ultrasonic extraction is selected as the extraction method for pretreatment of compounds.

[0240] 1.3.1.2 Optimization of Extraction Solvent

[0241] Compare the total ion chromatograms obtained under different extraction methods, such as Figure 2 When ethyl acetate was used as the extraction solvent, the peak separation was poor. When pure methanol was used as the extraction solvent, the number of peaks and the separation effect were good, with a high response intensity, but a significant solvent peak effect appeared after multiple injections. In contrast, when 75% methanol was used as the extraction solvent, the chromatographic peak separation effect and response intensity were both excellent. However, when 50% methanol was used as the extraction solvent, the chromatographic peak separation effect was poor, especially in positive ion mode (…). Figure 2In step A), almost no peak appeared after 24 minutes. Methanol, as a highly polar organic solvent, can effectively dissolve polar or semi-polar target components. A 75% methanol solution combines the high polarity of methanol with the polarity of water, possessing both good solubility and permeability. In contrast, pure methanol is too highly polar and may have limited solubility for certain components, while ethyl acetate has lower polarity and is more suitable for extracting non-polar or weakly polar components. Based on the above analysis, this experiment ultimately selected 75% methanol as the optimal extraction solvent.

[0242] 1.3.1.3 Optimization of Extraction Temperature

[0243] Compare the total ion chromatograms measured at different extraction temperatures, such as Figure 3 At 20℃, the number of peaks and the response intensity are both less than at 30℃. The response intensity at 40℃ is close to that at 30℃, but in positive ion mode ( Figure 3 (A) 14~20min and negative ion mode ( Figure 3 Within 16-20 minutes of step B), the number of chromatographic peaks decreased. This may be because the increased temperature caused the degradation of some thermally unstable compounds in the cigar tobacco leaves, resulting in a decrease in the number of peaks and a poorer extraction effect. Therefore, 30℃ was selected as the optimal extraction temperature for this experiment.

[0244] 1.3.1.4 Optimization of Extraction Time

[0245] Compare the total ion chromatograms obtained at different extraction times, such as Figure 4 Samples extracted for 30 minutes in positive ion mode (…) Figure 4 (A) 14~20min and negative ion mode ( Figure 4 In sample B), during the 16-20 min extraction phase, the number of chromatographic peaks was significantly higher than that of samples extracted at 20 min, 45 min, and 60 min. This may be because at 20 min, the compounds in the sample were not fully extracted, resulting in fewer peaks and lower response intensities. At 30 min, the compounds in the cigar tobacco leaves were fully extracted, resulting in the highest number of peaks and moderate response intensities. Further extending the extraction time reduced the number of peaks, indicating that excessively long extraction times can damage the stability of some compounds. Therefore, 30 min was chosen as the optimal extraction time for sample pretreatment.

[0246] 1.3.1.5 Optimization of the feed-liquid ratio

[0247] Total ion chromatograms obtained by comparing different feed-to-liquid ratios ( Figure 5 It was found that a feed-to-liquid ratio of 1:20 showed a significant advantage. In positive ion mode (… Figure 5In the 14-20 min range of phase A), the number of chromatographic peaks at a material-to-liquid ratio of 1:20 was significantly greater than that at 1:10; simultaneously, in negative ion mode (… Figure 5 In section B), the response intensity was also higher with a solid-liquid ratio of 1:20. Analysis showed that a solid-liquid ratio of 1:10, due to the excessively high extract concentration, might lead to insufficient extraction, thus affecting the extraction efficiency of the active ingredients; while a solid-liquid ratio of 1:20, by increasing the solvent volume, established a more favorable concentration gradient and improved mass transfer efficiency, thereby promoting the dissolution and diffusion of the active ingredients. Based on a comprehensive evaluation of chromatographic peak number, response intensity, and extraction efficiency, 1:20 was determined to be the optimal solid-liquid ratio.

[0248] 1.3.1.6 Optimization of purification conditions

[0249] Compare the total ion current chromatograms measured under different purification conditions, such as Figure 6 The poor peak count and response intensity after rotary evaporation are due to the loss of low-boiling-point and high-vapor-pressure volatile compounds during vacuum concentration; for example, some organic acids and alcohols may volatilize during solvent evaporation. Furthermore, rotary evaporation is a cumbersome and time-consuming process, which is not conducive to the subsequent extraction of large batches of samples. Therefore, considering various factors, centrifugation was chosen as the optimal purification condition.

[0250] 1.3.2 Optimization of chromatographic conditions

[0251] 1.3.2.1 Optimization of the mobile phase

[0252] By examining different mobile phase systems, such as Figure 7 Because cigar tobacco has a complex composition, containing a large amount of alkaloids and phenolic substances, peak tailing can be improved by adjusting the acidity of the mobile phase during reversed-phase chromatography. This is typically achieved by adding 0.1% formic acid. From the positive ion mode ion chromatogram (… Figure 7 As shown in A), the 0.1% formic acid-methanol system exhibits good separation but insufficient signal response intensity; the 0.1% formic acid-acetonitrile system shows poor peak separation in the first two minutes; while the 0.1% formic acid-0.1% formic acid-acetonitrile system not only demonstrates excellent peak separation and response intensity but also separates more compounds within 22-25 minutes. In the negative ion mode ion chromatogram ( Figure 7 In section B), the methanol-0.1% formic acid system showed a weaker signal response and fewer peaks, while the 0.1% formic acid-0.1% formic acid-acetonitrile system demonstrated significantly better separation performance than the 0.1% formic acid-acetonitrile system. Based on a comprehensive analysis of the performance under both positive and negative ion modes, this experiment ultimately determined the 0.1% formic acid-0.1% formic acid-acetonitrile system to be the optimal mobile phase.

[0253] 1.3.2.2 Optimization of Chromatographic Columns

[0254] from Figure 8 It can be seen that although the BEH C18 column and the HSS T3 column are similar in response intensity and number of peaks, there are significant differences in their separation performance. Positive ion mode ion chromatogram ( Figure 8 (A) shows that nicotine is not completely separated on the BEH C18 column, with a short interval between the two peaks; however, on the T3 column, the two optical isomers of nicotine are completely separated. Similarly, the negative ion mode ion chromatogram ( Figure 8 Figure B) shows that the T3 column also exhibits better separation performance for L-malic acid and 4-oxoproline, and the two compounds can be completely separated. Therefore, the HSS T3 column was selected for subsequent experiments in this study.

[0255] 1.3.2.3 Optimization of column temperature

[0256] By examining the column temperature of the chromatographic column, such as Figure 9 In positive ion mode ( Figure 9 In the (A) column, the number of peaks was lowest and the response was weakest at a column temperature of 20℃; column temperatures of 30℃ and 40℃ had similar effects on peak elution and separation. From the negative ion mode ( Figure 9 From the perspective of (B), column temperature has little effect on response intensity, peak number, and separation degree. Based on the significant improvement in response intensity in positive ion mode, and considering that the impact of column temperature increase on analytical stability is within a controllable range, this study ultimately determined 40℃ as the optimal column temperature condition.

[0257] 1.3.2.4 Optimization of Injection Volume

[0258] Through systematic investigation of three injection volumes of 0.5 μL, 1.0 μL, and 2.0 μL, such as... Figure 10 Different injection volumes have a significant impact on the analytical results. Positive ion mode ion chromatogram ( Figure 10 As shown in Figure A), when the injection volume is 0.5 μL, although the chromatographic peak separation is good, the low response intensity may lead to insufficient detection sensitivity for low-concentration compounds. Increasing the injection volume to 1.0 μL achieves a better balance between separation and response intensity. However, when the injection volume increases to 2.0 μL, although the response intensity significantly improves, the peak separation in the first two minutes deteriorates. (Negative ion mode ion chromatogram) Figure 10 A similar trend was observed in (B). Further research revealed that an injection volume of 2.0 μL caused oversaturation signals in the mass spectrometer detector due to high concentrations of compounds such as nicotine, leading to distorted quantitative results. Considering factors such as separation performance and detection sensitivity, an injection volume of 1.0 μL was the optimal choice.

[0259] 1.3.2.5 Flow rate optimization

[0260] By examining the three flow velocities, such as Figure 11 The results showed that flow rate had a significant impact on chromatographic separation efficiency and retention time. Positive ion mode ion chromatogram ( Figure 11 As shown in Figure A), at a flow rate of 0.2 mL / min, the chromatographic peak separation was good and the response intensity was moderate. When the flow rate was increased to 0.3 mL / min and 0.4 mL / min, the separation effect did not change significantly due to the shortened retention time of the sample components in the chromatographic column, but the peak width narrowed. (Negative ion mode ion chromatogram) Figure 11 B) shows the same trend. To ensure that most compounds have enough data points for chromatographic peaks without affecting chromatographic resolution, the liquid flow rate is set to 0.2 mL / min to moderately increase the peak width, thereby collecting more data points and avoiding the missed detection of low-content compounds due to insufficient data points.

[0261] 1.3.2.6 Optimization of Elution Gradient

[0262] Cigar tobacco has a complex composition. Gradient elution can gradually change the polarity or composition of the mobile phase, allowing components with different polarities or retention capacities to be eluted under suitable conditions, significantly improving separation efficiency. The optimized elution gradient results are shown below. Figure 12 As shown, elution gradient 1 exhibits the worst compound separation performance, with the lowest number of peaks and response intensity. Further optimization of gradient 2, from positive ion mode (…), further… Figure 12 As can be seen from A), the response intensity is enhanced, but the separation effect is not improved, especially in the negative ion mode. Figure 12 (B) Gradient 3 showed good separation performance in both positive and negative ion modes. However, during continuous sample analysis, a regular change in peak shape was observed in the first 4 minutes of the sample sequence, affecting compound separation. Gradient 4 improved upon the problems of Gradient 3 by shortening the pre-column equilibration time and mitigating the change in mobile phase gradient, but almost no chromatographic peaks appeared in the 4–12 minute range, resulting in poor overall separation performance. Gradient 5 showed good separation performance and high response intensity in both positive and negative ion modes; therefore, Gradient 5 was selected as the optimal elution gradient.

[0263] 1.3.3 Optimization of Mass Spectrometry Conditions

[0264] 1.3.3.1 Optimization of Collision Energy

[0265] High-energy collisional dissociation (HCD) is a crucial parameter in mass spectrometry. During a collision, ions convert their kinetic energy into internal vibrational energy. When this energy exceeds a certain threshold, the ions dissociate. In metabolomics, HCD can be used to identify and quantify small molecules by cleaving them and analyzing their fragment ions. However, higher collision energies are not always better. Excessively high energies can lead to over-fragmentation of some ions, while too low energies can result in the failure to fragment some ions, thus making compound identification unreliable.

[0266] Through optimization experiments on three collision energies, the results are as follows: Figure 13 As shown. When using lower energy E1 ( Figure 13 In step A), amino acid compounds failed to undergo effective fragmentation, resulting in a lack of fragment information for daughter ions. Energy E2 ( Figure 13 The setting of (B) is too high. Although it produces a large number of daughter ion fragments, the excessive fragmentation actually increases the risk of misidentification in compound identification. Energy E3 ( Figure 13 The uniform distribution of ion fragments generated by C significantly improves the accuracy of compound identification when performing matching with standard databases. Therefore, E3 is the most suitable collision energy parameter for this experimental system.

[0267] 1.3.3.2 Optimization of Acquisition Speed

[0268] To obtain more accurate identification results, the AquireX function, unique to the Orbitrap Exploris 480, was used. AquireX works by deeply characterizing scans, analyzing identifiable fragments in mass spectrometry data to discover more compounds, thus improving the efficiency and accuracy of analyte identification. The iterative precursor ion workflow creates an exclusion list based on HRAM MS analysis of a representative blank sample. AcquireX-DDA analysis is performed on the sample using the automatically updated exclusion list based on the selected precursor ions. This process can be repeated with other iterative sample analyses using the continuously updated exclusion list until a custom number of repeat injections is reached or no more precursor ions are available for analysis. When using AquireX, positive and negative ion modes are acquired separately, and the resulting positive and negative ion data are identified separately, avoiding additive ion identification errors during primary matching and resulting in more accurate identification results. During mass spectrometry acquisition, a slower acquisition speed increases the number of data points, ensuring sufficient data for each peak and improving peak integrity and reproducibility, but it also reduces the amount of compound information that can be extracted.

[0269] The results are shown in Table 6. First, background compounds were subtracted from parallel blank samples. Then, peak shapes were scored and screened, with peaks scoring ≥7 (i.e., smooth, symmetrical peaks without tailing or other anomalies) used as the screening criterion. Subsequently, compounds with secondary fragmentation were further screened, as secondary fragmentation can be matched with parent and daughter ion fragments in the compound library, significantly improving the accuracy of the identification results. The number of compounds in positive ion mode was greater than in negative ion mode, and the number of compounds with secondary fragmentation increased with increasing acquisition speed. Furthermore, as... Figure 14 As shown, when the acquisition speed is 10 scans / s ( Figure 14 In A), the number of data points for each peak is 18; while when the acquisition speed is increased to 15 scans / s ( Figure 14 In the B category, the number of data points was reduced to 14. To obtain high-quality data, a complete peak requires at least 10 data points. Therefore, a sampling rate of 15 scans / s not only meets the data point requirement but also allows for the discovery of more compounds. Based on the above analysis, this experiment ultimately determined 15 scans / s to be the optimal sampling rate.

[0270] Table 6. Compound Quantity Information at Different Acquisition Rates

[0271]

[0272] 1.4 Conclusion

[0273] The pretreatment conditions for cigar tobacco leaves were systematically investigated using single-factor experimental methods. Subsequently, based on the technical characteristics of chromatographic separation and mass spectrometry detection, key conditions such as chromatographic column, mobile phase composition, elution gradient, and collision energy were optimized to obtain the most complete and reliable analytical method system for the chemical components of cigar tobacco leaves. The results are as follows:

[0274] (1) The optimal pretreatment method for the sample is as follows: accurately weigh 0.500 g of cigar tobacco powder, place it in a 50 mL centrifuge tube, add 10 mL of 75% methanol, place the tube in an ultrasonic instrument, and extract it by ultrasonication at 30 °C for 30 min. After the extracted sample has cooled to room temperature, centrifuge it at 10000 r / min for 6 min, take the supernatant, filter it through a 0.22 μm microporous membrane, and set it aside for later use.

[0275] (2) Optimal chromatographic conditions: Column: Waters ACQUITY UPLC HSS T3 (100mm×2.1mm, 1.8μm); Column temperature: 40℃; Flow rate: 0.2mL / min; Mobile phase A: 0.1% formic acid water, Mobile phase B: 0.1% formic acid acetonitrile; Injection volume: 1μL. The elution gradient of mobile phase B was: -3-0min, 0%B (column equilibration); 0-4min, 0%B; 4-8min, 25%B; 8-14min, 65%B; 14-28min, 100%B; 28-32min, 100%B.

[0276] (3) Optimal mass spectrometry conditions: The ion source used was an H-ESI source; positive and negative ion detection modes were used, with a spray voltage of 3500V for positive ionization mode and 2500V for negative ionization mode; sheath gas flow rate was 40 Arb; auxiliary gas flow rate was 10 Arb; sweep gas flow rate was 0 Arb; ion transmission tube temperature was 320℃; vaporization chamber temperature was 275℃. The data acquisition time was 32 min, the primary mass number scan range was 100~1200 m / z; the primary full scan resolution was 60000; the secondary resolution was 60000; the RF setting of the S lens was 50%; the expected LC peak width was 6S; the acquisition speed was 15 scans / s; and the collision energy in MS / MS mode was 20, 40, and 80 eV.

[0277] Example 2: Identification of compounds in cigar tobacco leaves

[0278] This experiment aims to combine professional data analysis software with spectral analysis of existing databases to achieve high-throughput, high-accuracy, non-targeted rapid identification of cigar tobacco components and establish a database of compounds belonging to cigar tobacco.

[0279] 2.1 Experimental Materials and Equipment

[0280] Raw materials: Cigar tobacco leaves, originating from Deyang (SC-DY), Dazhou (SC-DZ), Lincang (YN-LC), Pu'er (YN-PE), Tunchang (HN-TC), Danzhou (HN-DZ), Enshi (HB-ES), Yichang (HB-YC), and Danjiangkou (HB-DJK) in Hubei Province. Reagents: Formic acid (chromatographic grade), Chengdu Kelong Chemical Co., Ltd.; chromatographic grade methanol, Thermo Fisher Scientific, USA; chromatographic grade acetonitrile, Thermo Fisher Scientific, USA.

[0281] Instruments and Equipment: JL-4500B Multifunctional Grinding Machine, Yongkang Sufeng Industry and Trade Co., Ltd.; SHB-Ⅲ Circulating Water Multipurpose Vacuum Pump, Zhengzhou Great Wall Science and Industry Trade Co., Ltd.; SB25-12DTD Ultrasonic Cleaner, Ningbo Xinzhi Biotechnology Co., Ltd.; HD-3000 Vortex Strainer, Hangzhou Youning Instrument Co., Ltd.; Milli-Q DIRECT 8 Ultrapure Water System, Shanghai Yixing Electromechanical Equipment Co., Ltd.; H17.5R High-Speed ​​Refrigerated Centrifuge, Shanghai Luxiangyi Centrifuge Instrument Co., Ltd.; HZY-224 / 323 Electronic Balance, Huazhi (Fujian) Electronic Technology Co., Ltd.; Vanquish™ UHPLC Ultra-High Performance Liquid Chromatograph, Thermo Fisher Scientific, USA; Orbitrap Exploris 480 High Resolution Mass Spectrometer, Thermo Fisher Scientific, USA.

[0282] 2.2 Experimental Methods

[0283] 2.2.1 Sample Pretreatment Method

[0284] Accurately weigh 0.500 g of cigar tobacco powder and place it in a 50 mL centrifuge tube. Add 10 mL of 75% methanol to each tube and place the tube in an ultrasonic apparatus. Extract the sample by ultrasonication at 30 °C for 30 min. After the extracted sample has cooled to room temperature, centrifuge it at 10000 r / min for 6 min. Collect the supernatant and filter it through a 0.22 μm microporous membrane for later use.

[0285] 2.2.2 Chromatographic and Mass Spectrometry Methods

[0286] Chromatographic conditions: Column: Waters ACQUITY UPLC HSS T3 (100 mm × 2.1 mm, 1.8 μm); Column temperature: 40℃; Flow rate: 0.2 mL / min; Mobile phase A: 0.1% formic acid in water; Mobile phase B: 0.1% formic acid in acetonitrile; Injection volume: 1 μL. The elution gradient of mobile phase B was: -3 to 0 min, 0% B (column equilibration); 0 to 4 min, 0% B; 4 to 8 min, 25% B; 8 to 14 min, 65% B; 14 to 28 min, 100% B; 28 to 32 min, 100% B.

[0287] Mass spectrometry conditions: H-ESI source was used; positive and negative ion detection modes were employed, with a spray voltage of 3500V for positive ionization and 2500V for negative ionization; sheath gas flow rate was 40 Arb; auxiliary gas flow rate was 10 Arb; sweep gas flow rate was 0 Arb; ion transmission tube temperature was 320℃; vaporization chamber temperature was 275℃. Data acquisition time was 32 min; primary mass number scan range was 100~1200 m / z; primary full scan resolution was 60000; secondary resolution was 60000; RF setting of the S-lens was 50%; expected LC peak width was 6S; acquisition speed was 15 scans / s; collision energies were 20, 40, and 80 eV in MS / MS mode.

[0288] 2.2.3 Compound Identification Procedure

[0289] (1) Select chromatographic peaks that are symmetrical, without forward extension or tailing, have a strong signal-to-noise ratio, and have a peak rating greater than 7.

[0290] (2) The qualitative analysis of mass spectrometry data needs to meet the 4-point rule. According to the requirements, a primary precursor ion in high-resolution mass spectrometry is worth 2 points, and a daughter ion in high-resolution mass spectrometry is worth 2.5 points. In the two secondary databases, MzCloud and MzVault, at least one precursor ion and one daughter ion need to be successfully matched to identify the compound.

[0291] (3) When the target compound in the sample matches multiple candidate compounds, the matching score between the secondary mass spectrum of the target compound and the secondary spectra of the candidate compounds in the spectrum library should be greater than 60 points. In addition, it is necessary to check whether the main secondary fragments can match the secondary fragments in the spectrum library one by one, and select the candidate compound with the best match as the target compound.

[0292] (4) For target compounds that have corresponding matches in the primary database and for which secondary spectra have been collected, but for which there are no matching secondary spectra in the secondary spectrum library, the chemical structure of the candidate compound needs to be theoretically fragmented. Based on the analytical matching of the secondary spectra after fragmentation, the matching degree should not be less than 60 points. If the major secondary fragments after fragmentation can be matched well, the matching score limit can be relaxed.

[0293] (5) The compounds that are on the edge of the screening criteria need to be included in the list of identified compounds in conjunction with literature analysis.

[0294] 2.3 Identification Results

[0295] like Figure 15 As shown, after identification and analysis, a total of 719 compounds were identified in 142 cigar tobacco leaf samples from 9 production areas, including amino acids, ketones, alkaloids, organic acids, phenols, nucleotides, lipids, sugars, terpenes, etc. Figure 15Among the compounds identified, organic acids were the most numerous, totaling 149, accounting for approximately 20.7% of the total. These mainly included nicotinic acid, succinic acid, and cinnamic acid. This was followed by amides (95 types) and terpenes (91 types), including typical components such as β-ionone, atractylodes glycoside A, and atractylodes lactone II. In addition, 75 lipid components and 73 phenolic substances were identified, such as coumarins, quercetin, chlorogenic acid, and rutin. 64 amino acid compounds were identified, including 20 common free amino acids as well as some modified free amino acids, such as methylated isoleucine and acetylated tryptophan and phenylalanine. 62 alkaloids were identified, covering components commonly found in cigar tobacco, such as nicotine, cotinine, nicotine, and their oxides. Other compounds included 36 ketones, 28 nucleotides, 16 sugars, 16 aldehydes, 8 alcohols, and 6 other compounds.

[0296] Example 3: Establishment of fingerprint profiles for cigar tobacco leaves and traceability of their origin

[0297] This study aims to construct a fingerprint map of cigar tobacco leaves by screening out differential characteristic markers among different origins and varieties; and to provide scientific basis and technical support for tracing the origin of cigar tobacco leaves through a random forest prediction model.

[0298] 3.1 Experimental Materials and Equipment

[0299] Same as item 2.1 in Example 2.

[0300] 3.2 Experimental Methods

[0301] 3.2.1 Sample pretreatment method

[0302] Same as item 2.2.1 in Example 2.

[0303] 3.2.2 Chromatographic and mass spectrometry conditions

[0304] Same as item 2.2.2 in Example 2.

[0305] 3.2.3 Data Processing and Analysis Methods

[0306] Non-targeted metabolomics detection was performed on the samples using the above methods to obtain total ion chromatograms for each sample. Freestyle 1.8 software (Thermo Fisher Scientific) was used to extract primary mass spectrometry extracted ion chromatograms (EIC) and peak areas for fingerprint similarity analysis. The extraction quality precision was set to 5 ppm, and the peak area integration parameters were: baseline window 80, peak height SNR 100, and peak area SNR 100. The extracted chromatograms and peak area information were exported as text document files. These text document files were then imported into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2012 Edition" software. One sample was selected as the reference chromatogram, and the mean method was used with a time window width of 0.1. Multi-point correction and full peak matching were performed to generate a control fingerprint chromatogram for domestic cigars.

[0307] 3.2.4 Fingerprint pattern establishment and evaluation methods

[0308] 3.2.4.1 Screening of Feature Markers

[0309] In the differential analysis of cigar tobacco leaves, characteristic compounds with large peak area differences, good repeatability, good chromatographic peak shape, high sensitivity, and low baseline interference were identified. Then, the chromatographic peaks of these compounds were extracted based on their precise mass numbers, and fingerprint spectrum similarity analysis was performed using extracted ion chromatograms (EIC) and their peak areas.

[0310] 3.2.4.2 Fingerprint Map Construction Method

[0311] The extracted chromatograms and peak area information of characteristic markers in the corresponding cigar tobacco samples were exported and imported into the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 version) software. One sample was selected as the reference chromatogram, and the mean method was used with a time window width of 0.1 to perform multi-point correction and full peak matching to generate a reference fingerprint chromatogram.

[0312] 3.2.4.3 Fingerprint Similarity Evaluation

[0313] Fingerprint similarity analysis is mainly calculated based on the retention time, peak height, intensity, and peak area of ​​chromatographic peaks. The similarity between each sample and the control fingerprint chromatogram was calculated using the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 version). This software uses the vector angle cosine method for similarity calculation.

[0314] 3.2.5 Constructing a Random Forest Prediction Model

[0315] The importance of compounds was ranked based on their VIP values, with higher rankings indicating greater importance. Data was used for training and testing according to modeling requirements, with separate training and testing sets. The training set was used to fit the prediction model, while the testing set was used to evaluate its performance. Key compound peak area data were divided into training and testing sets in a 7:3 ratio, and a random forest model was fitted using R. The training set data was imported for model training, and gradient boosting was used to fine-tune the model parameters. The testing set was used as an internal validation set to evaluate the performance of the fitted model.

[0316] 3.3 Results

[0317] 3.3.1 Characteristic markers

[0318] To avoid the influence of uneven sampling and systematic errors on peak area changes, compounds with good peak shapes and moderate corresponding intensities were selected. A total of 74 compounds were screened out through comparative analysis, as shown in Table 7.

[0319] Table 7. 74 Characteristic Markers in Cigar Tobacco Leaves

[0320]

[0321] Table 7 continues (1)

[0322]

[0323] Table 7 continues (2)

[0324]

[0325] Table 7 continues (3)

[0326]

[0327] 3.3.2 Cigar tobacco fingerprint spectrum

[0328] The chromatograms and peak area information of 142 cigar samples were exported to generate a control fingerprint (R). The results are shown below. Figure 16 .

[0329] 3.3.3 Random Forest Prediction Model

[0330] Unsupervised PCA analysis was performed on metabolites of cigar tobacco leaves from nine different origins to assess the overall metabolic differences and the magnitude of variability among samples. The results are as follows: Figure 17 In the PCA model, metabolites from the same origin clustered closely, while metabolites from some origins showed relatively clear distinctions. However, the distinctions between samples from Dazhou and Deyang in Sichuan, and between Enshi and Danjiangkou in Hubei, were not obvious.

[0331] Supervised LDA analysis was performed on all cigar tobacco leaf metabolites from nine origins. The samples were then classified using their discriminant functions. LDA discriminant analysis was conducted on cigar tobacco leaf samples from the nine origins, and LDA discriminant models were established for each origin. The results are as follows: Figure 18 In the LDA model, samples from Lincang, Yunnan, Pu'er, Yunnan, and other producing areas can be clearly distinguished using function 1, while function 2 can separate the nine producing areas.

[0332] PLS-DA analysis was performed on the metabolites of cigar tobacco leaves from nine different origins to screen for potentially differentially expressed metabolites. The PLS-DA model score plot ( Figure 19 As shown in Figure A), the samples within the same group are closely clustered, indicating good homogeneity. Meanwhile, except for the relatively dispersed distribution of samples from Pu'er, Yunnan, the other producing areas exhibit clear regional boundaries, suggesting significant differences in cigar tobacco metabolites among different producing areas. Samples from Deyang and Dazhou in Sichuan cluster together, and samples from Danjiangkou and Enshi in Hubei also highly overlap, indicating high metabolite similarity between samples from different producing areas within these two provinces. It is speculated that geographical proximity or similar planting conditions may lead to similar metabolic characteristics in tobacco leaves from these producing areas. Further PLS-DA analysis of the two groups showed complete separation. Figure 19 B, Figure 19 In the PLS-DA model, R... 2 Y=0.952, Q 2 =0.946, indicating that the established analytical model has good predictive and data interpretation capabilities.

[0333] The reliability and rationality of the PLS-DA model were examined using a 200-time random permutation test. Figure 20 ), Q 2 The intercept of the regression line is -0.249, which is less than 0.05, and the sample points are all located below the original points of the fitted line, indicating that the PLS-DA model has a good fit and there is no overfitting phenomenon.

[0334] Importance projection (VIP) reflects the strength of the influence of differences between different component groups on the classification of samples in the PLS-DA model. It can be used to initially screen components that have a significant impact on differences in origin, thereby identifying the components that differentiate between different origins. The top 200 compounds with the highest VIP values ​​were selected as the main markers of differences in cigar tobacco leaves from different origins (VIP=1.38).

[0335] The peak area data of 200 key compounds selected by the PLS-DA model were divided into training and test sets in a 7:3 ratio. The training set samples showed model training results, with a prediction success rate of 100%. The test set samples showed model testing results, with a prediction success rate of 100%. Therefore, this demonstrates that the random forest model has good classification prediction capabilities.

[0336] Table 8 Random Forest Training Results

[0337]

[0338] Table 9 Results of the Random Forest Test

[0339]

[0340] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0341] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. A method for constructing a fingerprint spectrum of cigar tobacco leaves, characterized in that, Includes the following steps: Cigar tobacco leaves were mixed with a solvent, subjected to ultrasonic extraction, solid-liquid separation, and the liquid was collected to prepare a test solution. The test solution was subjected to ultra-high performance liquid chromatography-tandem mass spectrometry to construct the fingerprint spectrum of the cigar tobacco leaves; The chromatographic conditions for the ultra-high performance liquid chromatography-tandem mass spectrometry include: the mobile phase comprises mobile phase A and mobile phase B. The mobile phase A is a 0.1% formic acid aqueous solution, and the mobile phase B is a 0.1% formic acid acetonitrile solution. The elution method is gradient elution. The elution gradient of the mobile phase is as follows: -3 min to 0 min, column equilibration, mobile phase B volume percentage 0%, mobile phase A volume percentage 100%; 0 min to 4 min, mobile phase B volume percentage 0%, mobile phase A volume percentage 100%; 4 min to 8 min, mobile phase B volume percentage changes from 0% to 25%, mobile phase A volume percentage changes from 100% to 75%; 8 min to 14 min, mobile phase B volume percentage changes from 25% to 65%, mobile phase A volume percentage changes from 75% to 35%; 14 min to 28 min, mobile phase B volume percentage changes from 65% to 100%, mobile phase A volume percentage changes from 35% to 0%; 28 min to 32 min, mobile phase B volume percentage 100%, mobile phase A volume percentage 0%.

2. The method for constructing fingerprint patterns of cigar tobacco leaves according to claim 1, characterized in that, The chromatographic conditions for the ultra-high performance liquid chromatography-tandem mass spectrometry include one or more of the following conditions: (1) The column temperature is 38℃~42℃; (2) The flow rate is 0.2 mL / min to 0.25 mL / min; (3) The injection volume is 1 μL to 1.5 μL; (4) The chromatographic column was Waters ACQUITY UPLC HSS T3.

3. The method for constructing fingerprint patterns of cigar tobacco leaves according to claim 1, characterized in that, The mass spectrometry conditions for the ultra-high performance liquid chromatography-tandem mass spectrometry include one or more of the following conditions: (1) The ion source is an H-ESI source; (2) Positive and negative ion detection modes are adopted. The spray voltage for positive ionization mode is 3000V~4000V, and the spray voltage for negative ionization mode is 2000V~3000V. (3) The collision gas is nitrogen; (4) The sheath gas is nitrogen, the sheath gas flow rate is 40 Arb~50 Arb, and the sheath gas temperature is 300℃~350℃; (5) The auxiliary gas is nitrogen, and the flow rate of the auxiliary gas is 10 Arb to 15 Arb; (6) The sweeping gas velocity is 0Arb; (7) The temperature of the ion transport tube is 300℃~350℃; (8) The temperature of the vaporization chamber is 250℃~300℃; (9) Data collection time is 30-35 minutes; (10) The scanning range of the first-order mass number is 100~1200 m / z; (11) The resolution of the first-level full scan is 60,000; (12) The secondary resolution is 60000; (13) The radio frequency voltage of the S lens is set to 50%~55%; (14) The expected LC peak width is 6S~7S; (15) Acquisition speed: 15 scans / s ~ 18 scans / s; (16) In MS / MS mode, the collision energy is set to 20eV, 40eV and 80eV respectively.

4. The method for constructing fingerprint profiles of cigar tobacco leaves according to any one of claims 1 to 3, characterized in that, One or more of the following conditions must be met: (1) The mass-to-volume ratio of the cigar tobacco leaves to the solvent is 0.5 g: (10~15) mL; (2) The solvent includes a methanol aqueous solution with a volume fraction of 70% to 80%; (3) The temperature for ultrasonic extraction is 25℃~35℃, and the extraction time is 30min~35min; (4) Solid-liquid separation is carried out by centrifugation, wherein the centrifugation is performed at 8000r / min~12000r / min for 5min~10min.

5. The method for constructing fingerprint patterns of cigar tobacco leaves according to any one of claims 1 to 3, characterized in that, The fingerprint spectrum has 74 characteristic peaks, and the substances corresponding to the characteristic peaks include: L-Threonic acid, allantoin, D-(-)-quinic acid, methylsuccinic acid, D-(+)-malic acid, isocitrate, succinic acid, pantothenic acid, acetylpropionic acid, pentenoic acid, caffeic acid, 3-methylsalicylic acid, azelaic acid, (15Z)-9,12,13-trihydroxy-15-octadecadienoic acid, 12-oxooctadecadienoic acid, arachidic acid, (±)-13-hydroxy-9,11-octadecadienoic acid, ethyl linolenic acid, 13(S)-hydroxy Octadectotrienoic acid, 16-hydroxyhexadecanoic acid, L-aspartic acid, L-valine, proline, N-acetyl-DL-glutamic acid, 4-oxoproline, L-aspartic acid benzyl ester, N-phenylacetyl-L-aspartic acid, N-acetyl-L-phenylalanine, indole-3-lactic acid, indole-3-acetyl-L-aspartic acid, N-acetyl-DL-tryptophan, tryptophan, DL-stachyine, trigonelline, (S)-nicotine Nicotine-N-oxide, N-methylnicotinamide, trans-3-indoleacrylic acid, cotinine, 5-methoxy-N,N-diisopropyltryptamine, lorinolide, asiatic acid, isostevimol, ligustrol A, perilla lactone, betulin, oleanolic acid, catechol, protocatechuic acid, aesculin, rutin, hyperoside, quercetin, kaempferol, choline, mannitol, 2-amino-1,3,4-octadecanetriol, β-carotene, L-glucose L-lactone, L-ribose-1,4-lactone, (3R)-4,4-dimethyl-2-oxotetrahydrofuran-3-ylβ-D-glucopyranoside, limonamide, aurantamide, docosahexaenoic acid ethanolamide, hexadecylamide, 1,7-bis(4-hydroxyphenyl)-5-methoxyheptane-3-one, phenylheptanone, hypoxanthine, xanthine nucleoside, p-coumaraldehyde, coenzyme Q2, porphyrin, 3-aminoquinoline, 8-hydroxyquinoline.

6. A method for detecting cigar tobacco leaves, characterized in that, Includes the following steps: The cigar tobacco leaves to be tested were mixed with a solvent and subjected to ultrasonic extraction to prepare the sample solution to be tested. The sample solution to be tested was detected by ultra-high performance liquid chromatography-tandem mass spectrometry to obtain a spectrum; The spectrum is compared and analyzed with the fingerprint spectrum of cigar tobacco constructed by the method described in any one of claims 1 to 5; The chromatographic conditions for the ultra-high performance liquid chromatography-tandem mass spectrometry include: the mobile phase comprises mobile phase A and mobile phase B. The mobile phase A is a 0.1% formic acid aqueous solution, and the mobile phase B is a 0.1% formic acid acetonitrile solution. The elution method is gradient elution. The elution gradient of mobile phase B is as follows: -3 min to 0 min, column equilibration, mobile phase B volume percentage is 0%; 0 min to 4 min, mobile phase B volume percentage is 0%; 4 min to 8 min, mobile phase B volume percentage changes from 0% to 25%; 8 min to 14 min, mobile phase B volume percentage changes from 25% to 65%; 14 min to 28 min, mobile phase B volume percentage changes from 65% to 100%; 28 min to 32 min, mobile phase B volume percentage is 100%.

7. The method for detecting cigar tobacco leaves according to claim 6, characterized in that, The chromatographic conditions for the ultra-high performance liquid chromatography-tandem mass spectrometry include one or more of the following conditions: (1) The column temperature is 38℃~42℃; (2) The flow rate is 0.2 mL / min to 0.25 mL / min; (3) The injection volume is 1 μL to 1.5 μL; (4) The chromatographic column was Waters ACQUITY UPLC HSS T3.

8. The method for detecting cigar tobacco leaves according to claim 6, characterized in that, The mass spectrometry conditions for the ultra-high performance liquid chromatography-tandem mass spectrometry include one or more of the following conditions: (1) The ion source is an H-ESI source; (2) Positive and negative ion detection modes are adopted. The spray voltage for positive ionization mode is 3000V~4000V, and the spray voltage for negative ionization mode is 2000V~3000V. (3) The collision gas is nitrogen; (4) The sheath gas is nitrogen, the sheath gas flow rate is 40 Arb~50 Arb, and the sheath gas temperature is 300℃~350℃; (5) The auxiliary gas is nitrogen, and the flow rate of the auxiliary gas is 10 Arb to 15 Arb; (6) The sweeping gas velocity is 0Arb; (7) The temperature of the ion transport tube is 300℃~350℃; (8) The temperature of the vaporization chamber is 250℃~300℃; (9) Data collection time is 30-35 minutes; (10) The scanning range of the first-order mass number is 100~1200 m / z; (11) The resolution of the first-level full scan is 60,000; (12) The secondary resolution is 60000; (13) The radio frequency voltage of the S lens is set to 50%~55%; (14) The expected LC peak width is 6S~7S; (15) Acquisition speed: 15 scans / s ~ 18 scans / s; (16) In MS / MS mode, the collision energy is set to 20eV, 40eV and 80eV respectively.

9. The method for detecting cigar tobacco leaves according to any one of claims 6 to 8, characterized in that, One or more of the following conditions must be met: (1) The mass-to-volume ratio of the cigar tobacco leaves to the solvent is 0.5 g: (10~15) mL; (2) The solvent includes a methanol aqueous solution with a volume fraction of 70% to 80%; (3) The temperature for ultrasonic extraction is 25℃~35℃, and the extraction time is 30min~35min; (4) Solid-liquid separation is carried out by centrifugation, wherein the centrifugation is performed at 8000r / min~12000r / min for 5min~10min.

10. The application of the fingerprint pattern construction method for cigar tobacco leaves according to any one of claims 1 to 5 or the detection method for cigar tobacco leaves according to any one of claims 6 to 9, characterized in that, It should include at least one of the following applications: (1) Application in tracing the origin of cigar tobacco leaves; (2) Application in the quality control of cigar tobacco leaves; (3) Application in the identification of cigar tobacco varieties.

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